Association of Avian Veterinarians

Comparative Examination of Testicular Biopsy Samples and Influence on Semen Characteristics

in Budgerigars (Melopsittacus undulatus)
Author(s): Maria Hänse, Volker Schmidt, Susanne Schneider, Angelique Della Volpe and Maria-
Elisabeth Krautwald-Junghanns
Source: Journal of Avian Medicine and Surgery, Vol. 22, No. 4 (Dec., 2008), pp. 300-309
Published by: Association of Avian Veterinarians
Stable URL: http://www.jstor.org/stable/30134215 .
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 Journalof Avian Medicineand Surgery22(4):300-309, 2008
c 2008 by the Association of Avian Veterinarians

Comparative Examination of Testicular Biopsy Samples

and Influence on Semen Characteristics in Budgerigars
(Melopsittacus undulatus)
Maria Hiinse, VetMed, Volker Schmidt, Dr MedVet, Dipl ECAMS, Susanne Schneider,
VetMed, Angelique DellaVolpe, VetMed, and Maria-Elisabeth Krautwald-Junghanns,
Prof Dr MedVet, Dipl ECAMS

Abstract: Although breeding of companion birds has become increasingly popular over the
last decades, evaluating avian fecundity is rarely addressed and remains a challenge. Whereas
histologic examination of a testicular biopsy sample is useful in evaluating reproductive status,
as well as in identifying pathologic changes, cytologic examination is a much quicker method
and is less invasive. To determine if cytologic examination of testicular impression smears is
reliable compared with histologic examination in assessing reproductive status, we evaluated
results in 16 live and in 14 dead male budgerigars (Melopsittacus undulatus).In most birds,
macroscopic evaluation, combined with histologic or cytologic examination, resulted in a
consistent grading of testicular reproductive stage. However, some testes assessed as inactive
macroscopically showed advanced reproductive stage on histologic examination. Although a
single biopsy caused no permanent damage to the testes and did not impact the general health
status of the examined birds, repeatedendoscopic examination resultedin testicularcicatrization
or adhesions with the air sacs. In addition, some birds had delayed recovery after endoscopy. A
biopsy of 1 testis done once or even repeatedly during a 1-year period had no impact on sperm
production or quality.
Key words: testis, testicularbiopsy, endoscopy, spermatologicalexamination, avian, budgerigar,
Melopsittacus undulatus

Introduction increased worldwide, evaluating fecundity of

Approximately one-third of all 355 psittacine
bird species are threatened with extinction world-
wide,' which causes immense concern among
conservationists. Breeding birds, either to propa-
gate frequently kept species or to preserve
endangered species, is complicated because of
factors such as seasonality and monogamy.
Therefore, knowing if a bird is in active repro-
ductive status is important. For decades, andro-
logic examination has played an important part in
assessing the reproductive status of mammals. In
poultry, the methods for andrologic examination
have also already been established.2 Although
breeding of psittacine birds has continuously
From the Clinic for Birds and Reptiles, An
Tierkliniken 17, 04103 Leipzig, Germany.
these birds has barely been acknowledged. Even
for avian veterinarians, assessing fertility remains
challenging, especially in male birds.3
To evaluate an individual bird's reproductive
status, an endoscopic examination is required.4
Although macroscopic examination of testes is
not reliable in all cases,5,6 histologic examination
of testicular biopsy samples provides the most
precise results.6'7 However, in a study of swift
parrots (Lathamus discolor), cytologic examina-
tion of testicular tissue was highly accurate for
testicular staging when compared with histologic
examination.6 Advantages of the cytologic meth-
od are the requirement of much less testicular
material and faster evaluation of samples.
In the 1980s, semen collection and analysis and
den artificial insemination were first described in a few
psittacine species, mainly budgerigars (Melopsit-

300

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 HANSE ET AL- TESTICULAR BIOPSIES IN BUDGERIGARS 301

Table 1. Stages of testicular cycle and cause of death in budgerigars(n = 14) during a 1-year study period.

Date of death Testicularstage Causeof death

06/27 Active Anorexia resulting in hypoglycemia because of crop impaction
07/28 Active Euthanasia because of traumaticallyinduced skull fracture
08/09 Transient Hypoglycemia because of enteritiscaused by Escherichiacoli; proventriculitis
caused by Macrorhabdusornithogaster;inclusion body hepatitis caused by
adenovirus
08/15 Active Polytetrafluoroethylene toxicosis
09/04 Active Euthanasia because of liver amyloidosis
09/20 Active Died during endoscopy, obesity
09/20 Active Died during endoscopy, obesity
12/07 Active Cerebrocorticalinjury
12/08 Inactive Squamous-cell carcinoma
01/19 Juvenile Cerebrocorticalinjury
03/16 Inactive Chlamydiosis
05/13 Transient Pneumonia granulomatosa mycoticans
05/16 Transient Fatty liver degeneration
05/21 Active Multiple renal cysts

tacus undulatus).8-12 Within the framework of Materials and Methods

these studies on psittacine semen, reference values
were established for budgerigars,8"1--c3cockatiels
(Nymphicus hollandicus),'4 monk parakeets
(Myiopsitta monachus),14 and, with some restric-
tions, peach-faced lovebirds (Agapornis roseicol-
lis).'4 In addition, by using systematic examina-
tion, spermatozoa that were morphologically
deviant were described for the first time in
Furthermore, semen collec-
psittacine species.14-'~'6
tion was repeatedly successful in individual males
of different psittacine species (Hispaniolan Ama-
zon parrots [Amazona ventralis], blue-naped
parrots [Tanygnathus lucionensis], and golden-
capped conures [Aratinga auricapilla])3,17 and
successful only once in others (blue-crowned
conures [Aratinga acuticaudata], Surinam brown-
throated conures [Aratinga pertinax surinam], sun
conures [Aratinga solstitialis], golden conures
[Guarouba guarouba], bronze-winged parrots
[Pionus chalcopterus], and red-tailed cockatoos
[Calyptorhynchus magnificus]). However, most
attempts of semen collection in larger parrots
were unsuccessful.' 7,"
The purpose of this study was to compare
the results of cytologic and histologic examina-
tion of testicular impression and biopsy samples,
respectively, with the macroscopic appearance of
the testis in determining the reproductive status
of male budgerigars. Furthermore, the develop-
ment of the individual reproductive cycle in
budgerigars was documented, and the influence
of endoscopic biopsy on the testes and fertility
was observed.
Birds
Dead birds: From June 2006 through May
2007, carcasses of male budgerigars (n = 14) were
examined within the scope of routine necropsy at
our clinic. All had either recently died or were
euthanatized because of various diseases, most of
which were noninfectious (11 of 14 budgerigars)
(Table 1). The testicular samples of these dead
birds were taken for later comparison with
endoscopically obtained samples.
Experimental birds. Sixteen male adult bud-
gerigars were purchased from breeders. Twelve
were bought in May 2006, and 4 were added to
the group in October 2006. Two female budger-
igars were kept together with the males for sexual
stimulation. All budgerigars were clinically
healthy and appeared in good nutritional condi-
tion. No parasites were detected in pooled
dropping samples. Triple swabs of conjunctiva,
choana, and cloaca, examined by using the
ClearView test (Unipath, Bedford, UK), were
negative for Chlamydophila species antigen in all
birds. Results of assays of pooled feather samples
tested by specific polymerase chain reaction'9,20
were negative for psittacine beak and feather
disease virus and avian polyomavirus.
During summer months, budgerigars were
housed in outdoor aviaries (3 x 1 x 2 m) and
exposed to environmental temperature and natu-
ral light (day length, 12 to 16 hours). From
November to April, they were kept in indoor
aviaries (2 x 1 x 2 m) with heating elements,

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302 JOURNAL OF AVIAN MEDICINE AND SURGERY

Table 2. Number of endoscopic examinations and
testicular biopsies in budgerigars (n -= 16) during a 1-
year study period.
Total birds/birds Birds
that died during undergoing Testicularbiopsy
Date
procedure (n) endoscopy (n) samples (n)
7/26/06 12/2 12
8/23/06 9 9
9/20/06 9/2 4a
11
1/24/07 1lb
4/03/07 11/1 11
a Endoscopy discontinued after 4 birds because of death of 2
birds.
b Four birds added to the study.
thermostats, ventilation, and lighting (daylight
and artificial light). The ambient temperature was
maintained at 150C by using a thermostat. Air
was circulated by negative-pressure ventilation.
The light cycle consisted of a light period of at
least 9 hours (ie, 9 hours artificial light plus time
of daylight) and a remaining dark period. The
birds were fed with mixed seeds ad libitum until
September 2006. Afterward, because of obesity
problems in some of the budgerigars, they were
put on a restricted diet, which consisted of 1
teaspoon of seeds twice daily for each bird. The
seed mix was provided in an individual feeding
dish for each bird and supplemented with fruits,
vegetables, salad, and herbs.
Collection of testicular samples
Dead birds: The testicles of necropsy speci-
mens were examined for color and consistency,
and the size of the testes in relation to adrenal
glands was determined. The reproductive status
was graded macroscopically,4 after which a
testicular sample was taken. An impression smear
of the sample was made and stained with
DiffQuik (Dade Behring, Marburg, Germany).
The remaining sample material was put into 4.5%
buffered formalin, then processed for histologic
slide preparation and stained with hematoxylin
and eosin.
Experimental birds. During a 1-year period,
endoscopic examination was performed 5 times in
2 budgerigars, 4 times in 5 budgerigars, 3 times in
2 budgerigars, twice in 4 budgerigars, and once in
3 budgerigars (Table 2). Endoscopy was done
immediately after sperm collection, except for 1
examination in the winter, when sperm was not
collected. For all procedures, birds were anesthe-
tized with isoflurane. Before each procedure,
birds were given lactated Ringer's solution with
5% dextrose (2:1; 20 ml/kg SC). The working
channel (4.8 mm), which contained the endoscope
(2.7-mm, 300, offset, rigid endoscope, Karl Storz,
Tuttlingen, Germany) was inserted into the left
flank cranial to the femur." The left testis was
investigated macroscopically, as described previ-
ously.4 A biopsy forceps with a diameter of
12 1.7 mm (Karl Storz) was introduced into the
8
working channel, and a testicular biopsy sample
1
was taken under visual control. Biopsy samples
7
10 were processed as described for testes obtained
from dead birds.
Cytologic examination
The slides stained with DiffQuik (Dade Behr-
ing) were examined by light microscope (BX41,
Olympus, Hamburg, Germany) by using x 1000
magnification (oil immersion). The structure and
activity of the germ cell samples were classified
into 4 stages: quiescence (inactive stage), sper-
matogonial multiplication (transitional stage),
spermatocyte division and elongation (active
stage), and regression and/or rehabilitation (also
classified as transitional stage) (Table 3).6
Histologic examination
Slides prepared with hematoxylin and eosin
stain were examined with x600 magnification.
The ratio of the tubule diameter to the surround-
ing interstitium and the number of tubular cell
layers were measured, the morphology of the
germ cells was described, and the existence of a
tubular lumen was assessed. For histologic
grading, samples were classified by the same
criteria as described for cytologic examination
(Table 3).6
Sperm collection and analysis
After 2 months of acclimatization, sperm was
collected from the budgerigars between July 2006
and July 2007, excluding fall and winter months
(mid October until the end of February). During
this time, the birds were exposed to nonstimula-
tory climatic conditions (cold, dry, short day
length), with the objective of inducing an inactive,
or at least less active, reproductive status. In
spring, 5 weeks after the fourth endoscopy, sperm
collection was recommenced. Semen was collected
weekly, in the morning, by using the massage
technique." Birds were physically restrained by an
assistant, and the cloaca was cleaned by using a
wet tissue. Afterward, the fore and middle fingers
of one hand were positioned on both sides of the

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 HANSE ET AL- TESTICULAR BIOPSIES IN BUDGERIGARS 303

Table 3. Stages of testicular cycle characterized by changes in cross-sectional appearance and architecture of
interstitial and tubular cells (modified from Gartrell's6scheme).

Testicularstage Spermatogoniacell structureand activity Tubulus/interstitium Tubular cell layers

Quiescence spermatogonia, large round pale nuclei, little T< I 1-2
cytoplasm, no cell division
Spermatogonial spermatogonia + spermatocytes, increased T I 2-6
multiplication cytoplasmic volume, active mitosis -
Spermatocyte division spermiogenesis, elongated spermatids present, T > I 6-10
and elongation active meiosis
Regression/rehabilitation smaller spermatogenetic cells, reduction in T > I 2-6
cytoplasmic volume, no cell division,
disintegrating spermatocytes
Abbreviations:T indicates tubulus; I, interstitium.

cloaca, while the thumb was positioned dorsally
at the tail base, and rhythmic cloacal massage was
carried out with gentle pressure.
After ejaculation, semen was collected in
calibrated microcapillary tubes (Wiretrol, 1 to
5 gl; Drummond Scientific Company, Broomal,
PA, USA). Semen volume was measured directly.
One part was applied to a pH indicator (pH-
Spezialindikator pH 6.5-10.0; Merck, Darmstadt,
Germany), and another part was applied to a
warm slide, mixed with 1 drop of 2% eosin, and
immediately smeared and dried. To determine the
dead-live ratio, 200 spermatozoa were counted by
using X400 magnification. In addition, 200
spermatozoa were assessed by using x 1000
magnification (oil immersion) for pathomorpho-
logic differentiation. If the volume was sufficient,
then the remaining sperm sample was diluted with
warm 1% glucose solution. One drop was applied
to a warm slide and covered with a cover slip for
immediate investigation of sperm motility by
x400 magnification. The remaining diluted sam-
ple was used to determine sperm concentration
and total sperm count with a Biirker-Tiirk
counting chamber (LO-Laboroptik, Friedrichs-
dorf, Germany).
Monitoring of health status
The birds were observed once daily for clinical
signs, in particular, weakness, reduced feed or
water intake, diarrhea, dyspnea, or other signs of
illness. The birds were examined clinically once a
week, and their body weights were measured
before sperm collection.21 During endoscopic
examination, the left testis was examined, and

Table 4. Comparison of macroscopic and cytologic (= histologic) stages from testicular biopsy samples in
budgerigarsduring a 1-year study period.

Stage
Active Transient Inactive
Macroscopic
Cytologic (=histologic) Active" Transientb Active" Transientb Transientb Inactivec
Month
Jun 1
Jul 12
Aug 9 1
Sep 4
Dec 1 1
Jan 4 1 2 2d
Mar 1
Apr 7 1 1
May 1 2
"Active stage: spermatocytedivision and elongation.
bTransientstage: spermatogonialmultiplication or regression/rehabilitation.
cInactive stage: quiescence.
dJuvenilestage of 1 testis of a 1-month-old budgerigar.

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304 JOURNAL OF AVIAN MEDICINE AND SURGERY

Figure 1. Active testis, samplingpoint in April. Endoscopic view of a left macroscopicallyactive testis (black arrow)
and of the biopsy forceps (white arrow) (top). Cytologic micrographof testis stained with DiffQuik (Dade Behring),
spermatozoa (black arrow); x 1000 magnification (left inset). Micrograph of testis stained with hematoxylin and
eosin, spermatozoa (black arrow); x400 magnification (right inset).

testicular cicatrization, adhesions, or permanent
holes in the air sacs were documented. After
recovery from anesthesia, the general condition of
each bird was evaluated. If necessary the bird was
hospitalized for monitoring or treatment until
rehabilitated.
Results
Comparison of macroscopic, histologic, and
cytologic examination
A total of 38 testicular biopsy samples from live
budgerigars and 14 testicular samples from dead
budgerigars were collected over a period of
1 year. The samples from dead budgerigars
included 2 deceased experimental birds. Sampling
was unsuccessful in 19.2% (9/47) of endoscopic
procedures, because the procedure was aborted
early or, in 1 case, the left testis was absent. Paired
samples were collected in all cases; however, in 18
of 38 biopsy samples (47.3%), a comparison could
not be done because biopsy samples were either
too small or too damaged for histologic exami-
nation. In contrast, all samples except one were
examined cytologically. In total, 37 of 38 biopsy
samples from live birds and 14 samples from dead
birds were evaluated by cytologic or histologic
examination. The macroscopic assessment was
erroneous compared with histologic or cytologic
results in 5 of 37 biopsy samples from live birds
(13.5%) and in 1 of 14 samples from dead birds
(7.1%). In 4 of those assessments (4 of 51 total
testicular samples [7.8%]), histologic results
showed a more advanced stage of spermatogen-
esis than suspected from the macroscopic appear-
ance (transitional or even inactive testis). In 2 of

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 HANSEET AL--TESTICULARBIOPSIESIN BUDGERIGARS 305

10 -
i0-
*

8-

r>

2--

07111 0726* 0~80 08~/23* 09/05 091201 10104 10/17 03106 04/16 I 0502 05114 0529 06113 0627
07119 0102 09/14 0c9/ 09/12 09/26 1009 02/27 0403* 04 24 0507 05/21 0605 06/11

Date of examination
Figure 2. Box plot diagram of sperm volumes obtained from budgerigars (n = 16) that underwent multiple
endoscopic procedures during a 1-year study period. O and * indicate single-bird statistical outliers; +, date
endoscopy was performed.

51 biopsy samples (3.9%), histologic examination
showed a transitional stage compared with an
active stage determined by macroscopic examina-
tion. Results of histologic examination revealed
an active reproductive stage in most samples (40
of 51 testicular samples [78.4%], Table 4). Of the
samples examined from carcasses, most birds (8
of 14) had active testes. Only 2 birds had inactive
testes in December and March. Three birds
showed transitional stages in May and August
(Table 1).
Of 7 biopsy samples collected in January from
experimental birds, 4 showed testicular regression
in the form of a transitional or even an inactive
stage in 1 case. On macroscopic examination in
April, 1 of the 7 birds, which had not been
examined the previous year, was in a transient
stage, whereas testes of the other 6 birds showed
an active stage in April (Fig 1). The testis of
another budgerigar was in a transitional stage in
April, ascertained on cytologic examination,
while the macroscopic appearance was still active
(Table 4).
Examination of sperm samples
Sperm parameters changed from summer to
winter. Average sperm production decreased at
the end of summer but did not stop entirely; in
some cases, endoscopic examination resulted in a
short temporary decrease in sperm production
and sperm quality. From March to July 2007,
most budgerigars produced smaller semen vol-
umes than in the previous year, accompanied by
lower sperm concentration and motility (Figs 2
through 4). However, the ratio of live spermato-
zoa (average, 86%-98%) in 2007 was similar to
that in 2006 (average, 93%-99%). Sperm pH
remained constant during the examination period
of 1 year (average, 8.1-9.0), whereas the quantity
of transparent ejaculates (8.1%) and blood
contaminated ejaculates (30.1%) was higher than

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306 JOURNAL OF AVIAN MEDICINE AND SURGERY

100-

80

60"

o-

0-

07111 07126" 08108 083" 095091 20* 10/04 10/17 03/06 04/16 05102 0529
07119 08/02 08114 s0829 09/b12 0926 10109 0227 0321 0424 05107
Date of exammnation
Figure 3. Box plot diagram of sperm motility obtained from budgerigars (n = 16) that underwent multiple
endoscopic proceduresduring a 1-yearstudy period. O indicates single-birdstatistical outlier; *, date endoscopy was
performed.

in the previous year (1.6% transparent ejaculates;
20.5% blood contaminated ejaculates).
Health status of the experimental birds and impact
of the endoscopic examination
In 71% of all endoscopic examinations, no
health or life-threatening complications occurred,
and most birds promptly recovered (Fig 5). In 4
birds, surgery was cancelled because of bleeding,
but these budgerigars recovered quickly. Howev-
er, 1 of the 4 birds showed this complication
during 3 consecutive endoscopic examinations.
Five budgerigars died because of severe hemor-
rhage during the endoscopic examination or the
next day. These birds were obese and were
possibly more prone to developing hemorrhage
than the other budgerigars.
During the second endoscopic examination, no
cicatrization or adhesions of the air sacs that
originated from the previous endoscopic proce-
dure were visible. During the third and fourth
endoscopy, minimal scarring was visible on the
left testes in 2 of 7 budgerigars. In 1 budgerigar,
the left testicle was no longer visible after the third
endoscopic procedure. In 4 birds, adhesions
between the air sacs and the left testis were
observed, with 1 showing both adhesions and
cicatrization. In many cases, the original entrance
through the abdominal air sac persisted and was
located during subsequent endoscopies.
Discussion
In this study, results of histologic and cytologic
examination of testicular samples from both dead
and live birds were equivalent. Cytologic exam-
ination proved to be a quick and valid method,
which required less testicular material than
histologic examination. Cytologic samples were
suitable for evaluation in almost all cases,
whereas the histologic sections could not always

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 HANSE ET AL--TESTICULAR BIOPSIES IN BUDGERIGARS 307

60-

50-

S40
o*
ar
QIx o

30

u0
u 0-

L1

20-

10808 oS(23*
07/1110726* 0915 10/170306 0416 050210514 09 10618
07/19 08/02 0814
09n5o*10t04
08329 09 12 09/26 10/09 0227 03121 04(24 05(21
05/07 06/05
Date of examination
Figure 4. Box plot diagram of sperm concentration obtained from budgerigars (n = 16) that underwent multiple
endoscopic procedures during a 1-year study period. O and * indicate single-bird statistical outliers; *, date
endoscopy was performed.

Results of endoscopies

40 et
8 35 mdeath
..
S30
.25 asurgeryabandoneddue to
.0 20
Shemorrhage
15
215 odelayed recovery

.,
10
E 5 Oquickrecovery

7/26 8/23 9/20 1/24 4/3 total

Date of endoscopy

Figure 5. Impact of endoscopy (n = 48) according to examination date and in total budgerigars that underwent
multiple endoscopic procedures during a 1-year study period.

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308 JOURNAL OF AVIAN MEDICINE AND SURGERY
Table 5. Reference values for sperm parameters in budgerigars.
Parameter Budgerigar"
Sperm volume (gl) 3.5-13.5
pH 8.2
Sperm concentration (X 106/Il) 9.5-11.3
Motility (%) n.s.
Ratio of live spermatozoa (%) n.s.
Abbreviation:n.s. indicates not specified.
be analyzed because of tissue damage or insuffi-
cient biopsy sample.
The reliability of macroscopic testicular inves-
tigation in these budgerigars was comparable with
that of previous studies: it is not valid in all cases,
particularly when macroscopic appearance is
indicative of inactive testes.6,7 However, the
location of testicular biopsy sampling is impor-
tant for the validity of examination results. If the
sample is taken too peripherally, then an insuf-
ficient number of tubule cross sections may be
obtained, thereby resulting in false results on
cytologic examination. This could explain the 2
histologic samples in this study from macroscop-
ically active testes that showed transitional stages.
In most cases, recovery after endoscopy was
uneventful. However, in small birds such as
budgerigars, there is inherent risk because of the
small size of the abdominal cavity. Obese
budgerigars had an increased risk of hemorrhage.
Nevertheless, if performed by an experienced
surgeon, testicular biopsy is a reliable and safe
method to evaluate the reproductive status or
testicular pathology, even in budgerigars. If done
once or twice, testicular biopsy seemed to have no
significant effect on sperm production or testicu-
lar morphology. However, many of the budger-
igars ejaculated sperm of inferior quality after a
winter rest period or did not ejaculate at all until
July 2007, although their testes were active and
they responded to cloacal massage. Minimal
cicatrization or, in 1 case, complete loss of 1
testis were seen in 3 budgerigars after the third
and fourth endoscopy. Furthermore, no impor-
tant changes in sperm quality could be detected
after the previous endoscopy in 2006. Other
factors, for example, changes in climatic condi-
tions, should also be considered as possible
triggers of reproductive status.22 During the first
6 months of 2007, rain and humidity were
abnormally low in Germany. This could be of
importance here, because testicular samples taken
during this period more often showed inactive or
transitional stages. In many parrot species, most
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Breeding-typebudgerigar'3 Exhibition-typebudgerigar'3
0.2-3.7 0.1-5.2
8.1-8.7 8.1-8.7
5.6-15.8 1.9-16.9
47-100 6-85
88-100 92-100
males cycle reproductively throughout the year.
Therefore, poor sperm quality or complete loss of
sperm production cannot automatically be attrib-
uted to a pathologic condition or general inability
to breed.
A potential negative influence of mixed housing
of sexes on dissemination results could be
excluded, because most results of sperm param-
eters of the budgerigars obtained in the second
half of 2006 reflected reference values established
in previous studies (Table 5).11c,"3 When male
budgerigars are housed separately, a dissemina-
tion interval of 7 days (unlike the natural mating
frequency in budgerigars of several copulations
per day) can result in an overaging of sperm.
Therefore, in groups of male budgerigars, a
dissemination interval of 2-3 days appears favor-
able regarding sperm quality.13 In conclusion,
endoscopically assisted testicular biopsy is an
adequate technique for assessing reproductive
status of male budgerigars with a negligible risk
on sperm production and quality.
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2008_red_listsummary_statistics/index.cfm. Ac-
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2. Schramm GP, Pingel H. Kiinstliche Besamung
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3. Brock MK. Semen collection and artificial insem-
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4. Clubb S. Reproductive assessment in psittacine
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5. Taylor M. Endoscopy, Practical lab. Proc Annu
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